Cells receive much of their information through signal transduction pathways that use GTP-binding regulatory proteins (G proteins) to convey signals from cell surface receptors to intracellular effector proteins. When these receptors, called G-protein-linked receptors, bind agonists, they promote the binding of GTP to specific G proteins. GTP binding activates the G protein and thereby allows it to regulate the activities of specific effector proteins.
The effectors regulated by G proteins include enzymes that synthesize cytoplasmic second messengers, ion channels, and transporters. G proteins are thus involved in regulating the synthesis and release of neurotransmitters, the sensitivity of synaptic receptors, general cellular metabolism, cellular differentiation, and growth.
The vital role of G-protein-linked receptors in enabling cells to respond to environmental signals explains their interest to basic researchers who wish to examine more closely their complex interactions with signals impinging upon the cell and with the G proteins which transduce that signal to effector pathways within the cell. It also explains the active interest of the pharmaceutical industry in applying the results of basic research to the development of drugs which interact with G-protein-linked receptors to affect basic cellular processes and treat human disease.
The identification of families of receptor subtypes has increased the need for inexpensive and facile assays for testing large numbers of potential drug candidates. Typically, radioisotopic ligand binding and second messenger assays are employed in screening drug candidates for their interaction with G-protein-linked receptors. These assay methods are difficult and time consuming to perform and involve the use of hazardous and expensive radioisotopes.
Others have proposed the use of chimeric receptors for testing potential drug candidates. This approach was applied to only one class of receptors, those having a single extracellular ligand-binding domain joined to a single intracellular domain which effects a change in the cytoplasm when the receptor binds ligand. In such receptors the two domains are often connected by means of a single highly hydrophobic region which embeds the receptor in a cell membrane. It is a simple matter to fuse the single extracellular region comprising the ligand binding domain to a heterologous reporter polypeptide, thus allegedly achieving useful chimeric proteins which effect an assayable response to ligand binding.
Many other receptors, including G-protein-linked receptors, have a more complex structure. In G-protein-linked receptors, for instance, ligand binding depends upon complex interactions among a number of different domains of the protein, including hydrophobic stretches passing through the membrane. To form chimeric receptors maintaining ligand specificity while effecting an easily assayed response requires a different approach than those previously described.
Thus there exists a need for a method which allows one to easily, cheaply and safely assay ligand binding to G-protein-linked receptors, providing a useful tool for drug discovery efforts, among others. The present invention fulfils these and other needs.